Bovine mucus penetration test and routine semen analysis of fresh and cryopreserved human spermatozoa

Semen samples from 18 fertile men and 18 infertile men were evaluated both fresh and after freezing and thawing using traditional semen parameter, the hypoosmotic swelling test (HOS-test) and the bovine cervical mucus penetration test (BMP-test). Major differences in the semen analysis between the fertile and infertile men were observed for motility, morphology (head and tail defects), the bovine mucus penetration test and the hypoosmotic swelling test. Cryopreservation resulted in a decrease regarding motility, the BMP-test and the HOS-test. Percentagewise the losses due to cryopreservation were higher in the infertile group than in the fertile group. The results of this study show that the bovine mucus penetration test as well as the HOS-test are valuable additions to the routine semen analysis of fresh and cryopreserved spermatozoa.     

Oocyte Penetration and Acrosome Reactions of Human Spermatozoa I: Influence of Incubation Time and Medium Composition

Washed sperm suspensions were evaluated for their ability to penetrate zona-free hamster oocytes and to exhibit an acrosome reaction in vitro. The percentage of acrosome reactions (AR%) was found to increase with incubation time and increased bovine serum albumin (BSA) concentration. We also found, however, that a remarkably high number of live, unreacted spermatozoa persisted during prolonged incubation in sperm samples obtained from some fertile men. After 22 hrs incubation, the motility of the spermatozoa was higher when the medium was supplemented with 10% human cord serum instead of 3.0% BSA. Penetration rates in hamster oocytes were not significantly different with 10% serum or 3.0% BSA as medium-supplementation. The addition of human instead of bovine serum albumin did not apparently affect the oocyte penetration rate of the spermatozoa.     

The influence of medium composition, osmolarity and albumin content on the acrosome reaction and fertilizing capacity of human spermatozoa: development of an improved zona-free hamster egg penetration test

The influence of medium composition, osmolarity and albumin concentration on the ability of human spermatozoa to undergo the acrosome reaction and penetrate zona-free hamster ova has been investigated. Raising the osmolarity but not the albumin concentration of the media was found to significantly increase the proportion of spermatozoa exhibiting an acrosome reaction and penetrating hamster ova, without influencing motility. There was, however, no correlation between the size of the acrosome reacted population and penetration rates between samples, suggesting that the zona-free hamster egg penetration test is more than just a measure of the availability of acrosome reacted cells. As a result of this study, a revised protocol for the hamster egg assay is described which is shorter and considerably more sensitive than conventional procedures.     

人精子顶体反应的检测及其与穿卵试验的相关性

选择正常生育男子（生育组）、输精管结扎再吻合（吻合组）、不育症患者（不育组）各２５例的精液进行人精子顶体蛋白酶及酸性磷酸酶测定,顶体组织化学三色染色和人精子穿透去透明带金黄仓鼠卵试验,比较不同生育条件下精子功能和生育能力的相关性。结果显示,精子获能后顶体反应率明显高于获能前（Ｐ＜０．０１）。生育组顶体反应率和穿透试验高于吻合组和不育组（Ｐ＜０．０１）。结果表明,精子穿卵试验结合精子顶体反应测定可作为客观评价人类精子受精能力的依据     

Assessment of hyperactivation, acrosome reaction and motility characteristics of spermatozoa from semen of men of proven fertility and unexplained infertility

Summary. Semen from men of proven fertility was compared with that of men with unexplained infertility to determine differences in spermatozoal functions such as hyperactivation and acrosome reaction and spermatozoal motility characteristics. The hyperactivated spermatozoa in both groups could be visualised on the monitor of the Computer Assisted Semen Analyser and they exhibited 'circling', 'thrashing', 'starspin' and 'helical' motility patterns and the mean hyperactivation rates were not significantly different. However, 20% of the men with unexplained infertility did not exhibit hyperactivation compared to only 4% in the fertile group. Furthermore, the semen from infertile men when evaluated for hyperactivation could be categorised into two groups with those having lower hyperactivation (<10% or <6% after 4 and 6 h of incubation respectively), forming the first group, and those having a higher hyperactivation rate constituting the second group. In the fertile men such distinct groups were not visible and the percentage hyperactivation ranged from 1 to 16%. No significant differences were observed in the rate of acrosome reaction of fertile and unexplained infertile men.
The non-hyperactivated spermatozoa from unexplained infertile men showed a significant increase in path velocity (VAP), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) and a decrease in linearity (LIN) and straightness (STR) compared to spermatozoa from fertile men. Furthermore, the hyperactivated spermatozoa from infertile men also showed an increase in progressive velocity (VSL) (only after 2 h of incubation) and LIN and decrease in ALH and beat cross frequency (BCF) compared to spermatozoa from fertile men. The results are discussed in the light of the importance of the above spermatozoal functions and spermatozoal parameters in fertilization.     

Diagnostic value of sperm function tests and routine semen analyses in fertile and infertile men

The results of routine semen analyses, the zona-free hamster oocyte penetration test, the hypoosmotic swelling test, and semen adenosine triphosphate levels were studied in 66 fertile and 130 infertile men. Multivariate discriminant analysis demonstrated that routine semen parameters including semen volume, sperm count, percent sperm motility, and percent normal spermatozoa in combination could predict the fertility of these patients with 70.4% accuracy. Of the three sperm function tests evaluated, the zona-free hamster oocyte penetration test and the hypoosmotic swelling test were selected by the multivariate discriminant analysis as variables capable of providing significant information on the fertility status of the patients. However, the addition of the results of these two tests to the routine semen analysis did not significantly improve the predictability of fertility. The overall correct prediction rate was 77.6% after incorporation of the results of these two sperm function tests. In this group of subjects, the presently available sperm function tests did not predict the fertility status of a patient with a high degree of accuracy.     

男性生育力的研究：三种精子功能测定方法的比较

应用精液常规分析(SFA),精子尾部低渗肿胀试验(HOS)和去透明带地鼠卵穿透试验(HOP)对15名能宵男性和15名不育患者的精液样本进行了综合检测和相关分析。能育组SFA参数异常者占20％,不育组SFA参数正常者占27％;精子尾部低渗肿胀率在能育组与不育组间无显著性差异(P>0.05);精子穿透率和受精指数在两组间差异非常显著(P<0.005,P<0.001)这表明HOP试验用于男性生育力的评价是一种较为准确,可靠的手段。本文还对各实验参数间的相关关系进行了分析和讨论。     

Correlation between human sperm swelling in hypoosmotic medium (hypoosmotic swelling test) and in vitro fertilization

Human ejaculates (n = 83) were analyzed for standard sperm parameters (concentration, motility, and morphology), as well as for the ability of the spermatozoa to react (swell) in a hypoosmotic medium (Jeyendran et al, 1984). Subsequently, the fertilizing capacity of the spermatozoa was tested by their ability to fertilize human oocytes in vitro. Although the sperm concentration was adjusted for in vitro fertilization, no adjustments were made for sperm motility and morphology. Correlation of the in vitro fertilizing capacity of the spermatozoa with the hypoosmotic swelling test (r = 0.56) was much higher than with standard sperm parameters (r varied from -0.04 to 0.25). Complete overlap was noted with standard semen parameters whether the ejaculate did or did not fertilize oocytes and ranged from very low to very high values in both cases. By contrast, all the semen samples that fertilized oocytes showed a 60% or higher reaction in the hypoosmotic swelling test, whereas the majority of the "infertile" semen samples showed less than 60% swelling. It therefore appears that, under the conditions of our studies, the hypoosmotic swelling test is a more accurate predictor of successful in vitro fertilization outcome than the conventional semen parameters. A combination of all parameters, however, is likely to be most useful. The hypoosmotic swelling test is simple and economical, and it is recommended that this test be further scrutinized for its value as an additional tool in the assessment of the in vivo fertilizing capacity of ejaculated spermatozoa.     

Adenosine 5''-Triphosphate (ATP) Concentration and Acrosin Activity in Human Spermatozoa. Their Relation with the Sperm Penetration Assay (SPA)

Fertility of men depends on the quality of semen. The aim of the present paper is to determine both the acrosin activity by radioimmunoassay and ATP concentration by bioluminescence in human spermatozoa, and evaluate these results in those samples with normal or low sperm penetration according to SPA test. Ejaculates obtained from 42 untreated men, were studied one hour after the obtention. These materials were divided into two groups:20 human semen samples with "in vitro" potentiality to penetrate zone-free hamster ova, between 15% to 98% and 22 human semen with SPA test between 0% to 14%. When we compare the group with normal penetration response vs that group with low or absent penetration ones, a significant decrease of ATP and acrosin concentrations was observed (P less than 0.001). Nevertheless no significant difference was observed in relation with percentage of motility, volume (ml), sperm concentration (10(6)/ml), percent of quick progressive spermatozoa and number of gametes capable of migrating into the medium layer (10(6)), between the group with low or absent penetration test against that one with normal zona-free hamster egg test.     

Assessment of sperm functions in infertile patients with varicoceles

Acrosin activity, acrosome reaction and nuclear chromatin condensation were studied in 24 infertile patients with varicoceles and 26 fertile men with or without varicocele. Chromatin condensation, assessed by aniline blue staining, and acrosin activity, evaluated by gelatinolysis technique, were significantly affected in the group of infertile patients. Defective chromatin condensation and defective acrosin activity were detected in 67% and 50% of the infertile patients, respectively. No significant difference was found between the two groups regarding the acrosome reaction, which was assessed by the triple staining technique after exposure of spermatozoa to low temperature (4 degrees C). This study identified a subgroup of infertile patients with normal standard semen parameters but impaired sperm functions. Results of the sperm function tests and standard semen parameters were not correlated. Therefore, it is concluded from this study that important sperm functions are impaired in patients with varicocele and that the gelatinolysis technique and aniline blue staining are effective tools for assessment of the fertilization potential of varicocele patients.     

Measurement of induced acrosome reactions in human sperm using physiologic stimuli--relevance for the prediction of fertilization outcome

Fertilization failure following standard in vitro fertilization in couples with normozoospermic men is an as yet unexplained phenomenon. A wide range of gametic disorders as well as environmental factors might contribute to this pathologic condition. One crucial condition appears to be the inability of the spermatozoa to undergo the acrosome reaction (AR). A discriminative test to distinguish fertile from non-fertile spermatozoa would be of utmost interest. In a prospective study, semen samples from men with normal semen parameters and fertilization failure were compared with semen samples from men with normal semen parameters and normal fertilization as to their capacity to undergo the AR. AR was induced using calcium ionophore as well as the physiologic stimuli progesterone and prostaglandin E(1). Discriminance analyses were undertaken to help identify patients with probable fertilization failure. Our data show that in patients with fertilization failure, the capacity of spermatozoa to undergo induced AR is greatly reduced using both unphysiologic and physiologic stimuli. However, physiologic stimuli are more suitable to identify patients with fertilization failure. Using physiologic stimuli, a formula was established to identify patients likely to fail at fertilization.     

A modified acrosome induction test

Many types of acrosome induction tests require special equipment and reagents that are not available to most clinicians; thus, simpler tests seem desirable. A modified acrosome induction test has been developed that uses basic reagents and a light microscope, which are available in most office settings. A hypoosmotic swelling test and a double stain (Bismark brown and rose Bengal) were combined to evaluate the viable acrosome reaction (AR) among 74 infertile men and 42 control men. The study included 34 infertile males without varicoceles, 20 with nonrepaired varicoceles and 20 with repaired varicoceles. On each test day, a specimen from a fertile donor was run as a control. The spontaneous acrosome reaction was recorded in semen before and after capacitation. The final % viable acrosome reaction equaled the capacitated value minus the spontaneous value for whole semen. The mean % viable AR among the control specimens was 16% with no values less than 10%. This mean value for controls was significantly greater than the mean % viable AR in each patient group. There were no overlaps in the 95% confidence intervals. When the study group was stratified according to normal acrosome induction tests or >10% viable AR, 30 patients had a normal test and 44 had abnormal tests. Six patients with varicoceles and an abnormal acrosome induction test had a varicocelectomy, and 2 (33%) converted their acrosome induction test to normal after at least 6 months of follow-up. Nine patients had in vitro fertilization (IVF), 3 had a poor result, and all had an abnormal acrosome induction test. Six had a good result with IVF and all 6 had a normal acrosome induction test. Thus, the acrosome induction test described in this report may be performed in any office laboratory to detect subtle male factor problems. The results may be helpful for planning IVF, intracytoplasmic sperm injection, or varicocele surgery for infertile men.     

Induction of the acrosome reaction in sperm by exposure to low temperature increases their rate of fusion with zona-free hamster oocytes

The fusion rate of human sperm with zona-free hamster ova was investigated after induction of the acrosome reaction by exposure to a low temperature (4 degrees C). Sperm were collected from 14 patients, and selected by the 'swim-up' method. The sperm were incubated for 24 h at either room temperature (control group) or at 4 degrees C (low temperature group), followed by additional incubation at 37 degrees C for 3 h. The mean sperm penetration rate, number of swollen sperm heads as well as the number of sperm attached to the oocyte increased significantly after exposing sperm to low temperature. The sperm penetration rate showed a significant correlation (Spearman test, r = 0.572, n = 28, P less than 0.0035) with the acrosome reaction in the low temperature group. These results were associated with an increase in the rate of penetration of hamster ova observed in this study, presumably due to the increase in induction of the acrosome reaction by low temperature. Incubation of sperm at low temperature might be useful in the evaluation of so-called false negative results in the zona-free hamster test.     

Relationship between hypoosmotic swelling test, semen analysis, and zona-free hamster ovum test

The hypoosmotic swelling test is a simple test for measuring the functional competence of the human sperm membrane. Fifty-four patients with idiopathic infertility were assessed by hypoosmotic swelling test and the results were compared with those of routine semen analysis and zona-free hamster ovum human sperm penetration test (ZSPT). Semen samples with abnormal semen parameters had lower percentage of swollen sperm in comparison with those with normal semen parameters. A positive correlation was observed between sperm concentration and sperm swelling (r = .50, p less than .05). A strong positive correlation was observed between the percentage of sperm motility and the percentage of swollen sperm (r = 0.60, p less than .01), and between motile sperm concentration and sperm swelling (r = .62, p less than .01). On the other hand, sperm swelling correlated only weakly with the percentage of sperm penetration. The results indicate that the hypoosmotic swelling test appears to evaluate different functional qualities of sperm than ZSPT.     

Correlation of the hypo-osmotic swelling test, semen score and the zona-free hamster egg penetration assay in humans

The correlation between the hypo-osmotic swelling test, the modified Eliasson score for human semen analysis and the hamster egg penetration assay was examined. The results showed a weak but significant correlation between the group of subjects with swelling rates below 50% and the above 80% group. These groups showed hamster egg penetration assay rates of 9 +/- 14% and 39 +/- 29%, respectively (P less than 0.035). A significant correlation was also found between modified Eliasson scores (17 +/- 10 and 5 +/- 7, respectively) and swelling rates below 50% and above 70% (P less than 0.001). These results suggest that spermatozoa showing a low swelling rate have a poor fertilizing capacity. Therefore, the swelling tests and analysis of the patients semen can be used to discriminate sperm quality.     

不同获能时相对人精子膜完整性的影响

本研究用人精子低渗肿胀试验（ＨＯＳ－ｔｅｓｔ）分析了１２例正常育龄男子的精液标本。精子分别用常规洗涤法、上游法和加入钙离子载体（Ａ２３１８７）诱导获能等三种不同方法处理后，分别在液化后、上游１ｈ后、获能３ｈ、６ｈ、２４ｈ后测定精子的低渗肿胀率，结果显示经不同方法处理、在不同获能时相之后的精子，在２４ｈ之内其肿胀率保持在８７．８±１４．６％～７３．３±１１．９％之间，统计学分析表明各标本组肿胀率之间无明显差异。本研究提示在２４ｈ内精子膜的完整性不受获能时相及不同处理方法的影响。     

The Kinetic of Acrosome Reaction – an Additional Sperm Parameter?

Spermatozoa of six men with normozoospermia, oligozoospermia and teratozoospermia were capacitated in vitro in modified Tyrode's medium und evaluated for acrosome reaction using the recently described triple-stain technique. The resulting kinetics illustrate the different capacitation behaviour and ability of spermatozoa to undergo acrosome reaction. The data were completed by the movement characteristics of spermatozoa during the incubation period. The kinetic of acrosome reaction seems to be an important functional sperm parameter that may be useful to discriminate between fertile and infertile spermatozoa.     

Assessment of sperm function in fertile and infertile men

Summary. The sperm function of fertile men (control), infertility patients (experimental), and men with varicocele were compared. The bioassays used were the follicular fluid-induced acrosome reaction, the binding to the zona pellucida, and the penetration of zona-free hamster oocytes. The percentage (mean ± SEM) of reacted spermatozoa was 35 ± 3 in the control, 22±1 in the experimental, and 22 ± 3 in the varicocele. The minimum value of acrosome reaction in control men was 20%. The mean number of zona-bound spermatozoa was 250 ± 30 in the control, 160 ± 28 in the experimental, and 196 ± 44 in the varicocele. The minimum number of zona bound spermatozoa in control men was 50. The mean number of hamster oocytes penetrated was 50 ± 8 in the control, 19 ± 3% in the experimental, and 10 ± 3 in the varicocele. The minimum number of oocytes penetrated in control men was 6%. In the experimental group, 22 men had a normal sperm function, 58 had 1 or 2 bioassays below the minimum (relative dysfunction), and 10 had all bioassay below the minimum (abnormal sperm function). The results of these bioassays could help to reclassify the infertile men in several subgroups.     

Reappraisal of the hypo-osmotic swelling test to improve assessment of seminal fertility status

The hypo-osmotic swelling (HOS) test has been proposed as a useful assay for evaluation of the functional competence of the human sperm membranes. To assess this further, the HOS-test was evaluated in 187 semen samples collected from fertile men and from male patients consulting for infertility. These samples were classified as normal, oligo-, astheno- or oligoasthenozoospermic on the basis of their standard semen variables. The percentage of total sperm tail swelling and of sperm exhibiting different tail swelling patterns was recorded. In the fertile men and in the group of patients with normal semen variables, significantly more (P less than 0.001) HOS-reactive sperm were observed after hypo-osmotic treatment in comparison with those groups exhibiting abnormal semen parameters. Swelling of the sperm in a hypo-osmotic medium was highly correlated with both progressive motility (r = 0.62, P less than 0.001) and sperm viability (r = 0.65, P less than 0.001). A weak positive correlation was also observed between sperm swelling and sperm morphological features (r = 0.31, P less than 0.005) and between sperm swelling and sperm concentration (r = 0.31, P less than 0.005). No significant correlation was observed between sperm swelling and in-vitro sperm fertilizing capacity as assessed by the zona-free hamster oocyte penetration assay. However the majority of the semen samples (87.3%) showing a normal penetration rate (greater than or equal to 10%) also exhibited a 60% (or higher) reaction in the HOS-test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does the hypoosmotic swelling test predict human sperm viability?

Human sperm viability is essential for successful fertilization. Eosin Y is the usually accepted method for sperm viability assessment, though the hypoosmotic swelling test has been proposed for the selection of viable spermatozoa in procedures such as intracytoplasmic sperm injection. The present study was designed to determine the value of hypoosmotic swelling test in the prediction of sperm viability. For this purpose, hypoosmotic swelling and eosin Y were performed in parallel and in combination, on both fresh and freeze-thawed semen. Rates for eosin Y were significantly higher than for the hypoosmotic swelling test in fresh semen, with a weak, though significant correlation between the two tests (r = 0.47, p < 0.05). When both tests were performed in succession (hypoosmotic swelling test followed by eosin Y), 14.6% of swollen sperm incorporated the dye. Following exposure to hypoosmotic conditions, sperm viability decreased by 35%. When sperm were killed by freezing, hypoosmotic swelling test rates were higher than eosin Y. Results indicate that these two tests cannot be used interchangeably, since 15% of the swollen sperm apparently died, suggesting that plasma membrane integrity is lost before the capacity to maintain osmotic equilibrium.